3 years ago

Conditional Displacement Hybridization Assay for Multiple SNP Phasing

Conditional Displacement Hybridization Assay for Multiple SNP Phasing
Tsz Wing Fan, I-Ming Hsing, Henson L. Lee Yu
The two chromosomal copies of the human genome are highly polymorphic, and the allelic content on each strand can dictate a person’s biological outcomes. While many of the current diagnostic tools are able to detect the presence of multiple mutations at the same time, most cannot determine the phase of these mutations unless long-range PCR or sequencing techniques are used or if templates are compartmentalized into single copies prior to amplification. Here, an enzyme-coupled hybridization assay, named conditional displacement hybridization assay (CDHA), is described for the concurrent and rapid determination of the presence and phase of SNP variants. In this approach, short DNA probes were utilized to first quantify the amount of SNPs on the templates using a two-channel fluorescence measurement. The hybrids formed between the probes and the templates then set up the right condition for the subsequent enzymatic displacement and quenching of a fluorophore-labeled strand, which happens only if both SNPs are present on the same strand. The drop in the fluorescence signal thereby indicates the phase of the two SNPs. As a proof of concept, we tested the assay on four variants of an arbitrary sequence—with or without mutation on two sites 100 nts apart. The assay described herein was able to determine the haplotype phase of the samples in less than 1 h. This method promises a direct, cost-effective, and laboratory-based test to extract further genetic information to determine and/or predict diseases and traits dependent on SNP phasing.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b02300

DOI: 10.1021/acs.analchem.7b02300

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